Passage of ribosomes through microsprayer increases functional activity – Implications for activity assays in time-resolved cryo-EM

Journal of Structural Biology · 2025-07-04

Calculation of specificity constants Γ for RUBISCO of different species from microcalorimetric data

Photosynthesis Research · 2025-12-01

Abstract Based on earlier data concerning the reaction enthalpy $$\:{\varDelta\:}_{\text{r}}H$$ of Ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO) from spinach (Frank et al. Phys Chem Chem Phys 2:1301–1304, 2000), it is shown that the specificity constant $$\:{\Gamma\:}$$ , indicating the ability of RUBISCO to discriminate between CO 2 and O 2 as substrate, can be determined from an isothermal titration calorimetric (ITC) measurement of $$\:{\varDelta\:}_{\text{r}}H$$ as a function of the substrate concentration ratio $$\:\rho\:=\left[\text{C}{\text{O}}_{2}\right]/\left[{\text{O}}_{2}\right]$$ . The approach does not need any radioactive materials and might be a cost-effective alternative for RUBISCO engineering to screen for variants with a high specificity constant.

BPS2025 - Structural and functional investigation of ryanodine receptor 1 with endogenous ligands and drugs

Biophysical Journal · 2025-02-01

283 Hierarchical protein unfolding at the site of a functional conformational change in human CFTR is the Achilles’ Heel controlling biogenesis efficiency and corrector-drug mechanism

Journal of Cystic Fibrosis · 2025-10-01

The mechanism of ribosomal recruitment during translation initiation on Type 2 IRESs

bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-11 · 1 citations

The encephalomyocarditis virus (EMCV) IRES and other Type 2 IRESs comprise domains H-L and specifically interact with eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES function, which also requires the preceding domain I of unknown function. To identify interactions that drive ribosomal recruitment of eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. It revealed that the apical domain I cloverleaf contacts ribosomal proteins uS13 and uS19 via its Id subdomain and that the essential GNRA tetraloop in subdomain Ic interacts directly with the TψC domain of initiator tRNA. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of primary and secondary structures of the apex of domain I among Type 2 IRESs suggests that the reported interactions are a common essential feature of them all.

Calculation of specifity constants Γ for RUBISCO of different species from microcalorimetric data

Research Square · 2025-08-29

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering

RNA · 2025-03-06 · 1 citations

(rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single-nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institute's rabbit whole-genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

A PDMS-based Microfluidic Chip Assembly for Time-Resolved Cryo-EM (TRCEM) Sample Preparation

BIO-PROTOCOL · 2025-01-01 · 1 citations

coating on the PDMS surface and fabrication of the microfluidic chip assembly. • Preparation of time-resolved cryo-EM sample in the time range of 10-1,000 ms. • Data collection on EM grid covered with droplets from the microsprayer.

Structural Studies of Nedicistrovirus IRES-Driven, Initiation Factor-independent Translation Shed Light on Key Steps of Eukaryotic Translation Elongation

bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-29

We utilized the Nedicistrovirus (NediV) intergenic region (IGR) IRES-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl-tRNA-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound post-proofreading complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading.

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering

bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-12 · 1 citations

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institute's rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.